2. INTRODUCTION
• The immune system in general responds appropriately to the
presence of foreign antigens.
• However, there are certain diseases that arise from either a defective
or overresponsive immune system on the part of the host.
• Two major therapeutic approaches are possible: either
immunosuppression or immunopotentiation of the immune system
4. Immunosuppressive Drugs
Corticosteroids
• Modulate inflammation by suppressing cytokine- and chemokine-
encoding genes, which inhibits the activation and recruitment of
inflammatory cells
• The side effects of steroids are numerous and often depend on both
the dose used and duration of treatment.
• These include an increased susceptibility to infection, osteoporosis,
and growth disturbances in children, as well as gastric ulcers,
hypertension, acne, and hirsutism
5. Immunosuppressive Drugs
• The development of the thiopurines in the 1950s ushered in a new
group of immunosuppressive agents, the most important of them
being azathioprine
• It is inactive until it is metabolized in the liver and takes three to four
weeks to be effective.
• The metabolites work by inhibiting DNA synthesis in dividing cells
(such as activated lymphocytes).
• Like many other drugs, it has side effects, mainly in bone marrow
toxicity, and long-term use eventually results in granulocytopenia and
thrombocytopenia
6. Immunosuppressive Drugs
• Another group is the alkylating agents, of which cyclophosphamide is one
of the best examples.
• This drug also requires activation by the liver. It inhibits cell division and
can suppress antibody production, and it decreases delayed-type
hypersensitivity
• Methotrexate, which inhibits cell division by disrupting folic acid
metabolism, has similar immunomodulatory effects
• Cyclosporin, a naturally occurring fungal metabolite, also inhibits T-cell
activation and cell-mediated immunity
• Tacolimus is a newer-generation drug with a similar mechanism of action.
7. OTHER IMMUNOSUPPRESSIVE METHODS
• There are a number of instances in which antibodies have been used
to suppress the immune response
• Among the earliest measures has been the use of anti-RHD antibodies
• More recently, monoclonal antibodies are being used to suppress the
immune system
• Antibodies that target the immune system can target cell surface
molecules on T or B cells or can target soluble mediators of
inflammation such as cytokines
• Other methods of immunosuppression are plasmapheresis or plasma
exchange
8. IMMUNOPOTENTIATION
• Chronic infections such as HIV and hepatitis C are characterized by an
inability of the host to control viral replication
• The ability to potentiate the host immune response to control chronic
infections is an important goal and is under active investigation.
• At least some types of cancer can be controlled by the host immune
system, so potentiation of the host immune system may prove useful
in treating these cancers
• There are three principal ways to potentiate the immune response in
humans: through cytokines, adoptive immunotherapy, or vaccination
9. IMMUNITY
• The two ways to achieve immunity are actively and passively
• Active immunity is achieved when exposure to a foreign stimulus triggers
an immunological response to the agent by the host
• Artificial active immunization is the administration of an immunogen as a
vaccine
• Vaccines may be live organisms, killed organisms, or modified toxins.
• Although no vaccine is ideal and each has its problems
10. CYTOKINE IMMUNOMODULATION
• There is increasing interest in targeting T cells or the cytokine
regulators to regulate the elimination of autoreactive T cells, tumor
cells, and the maintenance of a specific memory response to these
pathogens
• Such immune responses are normally regulated by cytokines, and the
activities of the cytokines have a high degree of redundancy.
• There are several distinct differences in the functions of IL-2 and IL-5.
IL-2 is involved in checkpoints or brakes on the immune system.
12. Immunological Techniques
• Laboratory tests vary widely in clinical immunology.
• Some are essential for diagnosis while others are useful in subclassifying
disorders
• These tests do vary in their sensitivity and specificity.
• Some assays are quantitative in that they produce precise results
• Qualitative assays are less specific and will give answers such as normal–
abnormal, or positive–negative results
13. ANTIBODY PRODUCTION
• Antibodies for various tests can be produced in a number of different
ways
A. Polyclonal antibodies: Many mammals have been used to produce
antibodies
B. Monoclonal antibodies
14. IMMUNOLOGICAL ASSAYS
• The introduction of automated machines to measure
immunoglobulins and other proteins has proceeded rapidly in recent
decades.
• Precise measurement of serum immunoglobulins is an essential
cornerstone in this area and is important for repeated and serious
infections
• The main principle behind this test is related to the formation of
immune complexes between the antibody and a given antigen.
• These tests primarily use polyclonal antibodies for each antigen since
monoclonal antibodies do not form immune precipitates because
there are too few relevant epitopes
15. Radioimmunoassay and Enzyme Linked
Immunosorbent Assays (ELISAs)
• The use of these highly sensitive assays in human disease has virtually
exploded in the past two decades
• They can be used to detect the levels of a given antibody or hormone
in human serum, and they are extremely sensitive methods of
detecting low levels of autoantibodies.
• Once the serum or purified antibody or antigen to be tested is placed
in the well, a second radio labeled antihuman IgG antibody is placed
in the well
• After appropriate binding and further washes, the degree of activity
of the antibody to a given antigen can be determined
16. Complement Assays
• Perhaps the most useful assays for complement are the
immunochemical assays of C3 and C4.
• The alternative and classical modes of complement breakdown, a low
C3 and C4 but normal factor B suggest that activation of the classical
pathways has occurred
• Examples would be patients with systemic lupus erythematosus or
vasculitis
• In contrast, if C3, C4, and factor B are all low, the alternative pathway
is also activated via either feedback loops or simultaneous activation
• This would point to a gram negative bacteremia.
19. DNA ANALYSIS
• Known unique segments of nucleic acid sequences can be used as
DNA probes to determine the presence of complementary sequences
of DNA in a sample from a given patient
• The probe, which is a single strand of a given DNA, is presented to the
target DNA, which is composed of thousands of nucleotides
• The complementary strands from target and probe DNA will anneal to
each other, a process known as DNA hybridization
• The high affinity of the probe for a complementary segment in the
target DNA is the most specific intermolecular interaction between
biological macromolecules
Among the oldest of these drugs are the corticosteroids, which have long been known to alter immune responses.
When corticosteroids are given, the result is a transient lymphopenia peaking at four hours and lasting up to twenty-four hours.
Helper T cells are predominantly affected, and at higher doses of steroids inhibition of interleukin-2 (IL-2) production by helper T cells becomes increasing important
Another major effect in humans is on resting macrophages (activated macrophages are not sensitive)
In humans, steroids are used for two main purposes. One is the prevention or reversal of graft rejection
The other is in the treatment of autoimmune and malignant diseases
Cyclosporin:The drug becomes active only when complexed to its intracellular receptor cyclophilin, and it inhibits early calcium-dependent events, especially the activation of several cytokine genes.
Its major effect is the inhibition of IL-2 production and the CD4+ proliferation responses. Cyclosporin has been extremely useful in the control of transplant rejection and is also used in several autoimmune diseases such as psoriasis and severe rheumatoid arthritis
However, long-term use has demonstrated severe toxicity such as nephrotoxicity and hepatotoxicity and particularly lymphoma induction.
several antibodies are now approved for the treatment of autoimmune diseases
These antibodies are typically “humanized” mouse monoclonals, created by transposing the mouse antigen-binding sites onto a human antibody framework
This technique retains the full range of effective properties of human Fc while minimizing the immunogenicity of the mouse component
Among the most effective uses of monoclonal antibodies has been in treating severe rheumatoid arthritis, using monoclonal antibody directed against tumor necrosis factor (TNF-α)
The drawback to this therapy is that the infusions must be repeated frequently to sustain results
Monoclonal antibodies can also be used as antitumor agents
Specific targeting and killing of tumor cells can be enhanced by linking tumor antigenspecific monoclonal antibodies to agents as follows:
a cytotoxic drug such as methotrexate,
a radioisotope such as iodine131 or ytrium-90, or (3) a toxin such as ricin.
Monoclonal antibodies are now approved for the treatment of non-Hodgkin’s lymphoma, myeloid and lymphocytic leukemia, breast cancer, and colorectal cancer
Total lymphoid irradiation produces longterm suppression of helper T cells and has been used in some severe autoimmune diseases like lupus or rheumatoid arthritis.
The side effects of this treatment may be severe and sometimes fatal.
Cytokine Therapy Interferons are antiviral glycoproteins, which are secreted as a result of a viral infection and have wide-ranging antitumor and immunomodulatory effects. They have attracted much interest as immunotherapeutic agents.
Interferons bind to cell surface receptors and activate secondary intracellular changes which inhibit viral replication.
They can be divided into three groups: alpha (α), beta (β), and gamma(γ) interferons
IFN-α is the treatment of choice for hepatitis B and C; when given systemically, it produces significant clearing of hepatitis B in chronic carriers.
IFN-α has some side effects, mainly flu-like symptoms such as fever, malaise, and anorexia – all symptoms that can be tolerated.
More severe effects are reversible: bone marrow depression, liver dysfunction, and cardiotoxicity
IFN-β has been shown to be of benefit in patients with relapsing-remitting multiple sclerosis, and IFN-β1 appears to decrease the rate of progression of disability.
Despite these results, the precise therapeutic role of IFN-β is still controversial.
IFN-γ is a potential activator of macrophages and is most often used in conditions in which defective macrophage function occurs.
Examples of these disorders are lepromatous leprosy, leishmaniasis, and chronic granulomatous disease.
IFN-γ works by increasing phagocytic bactericidal activity, but only some patients show enhanced superoxide activity, implying that IFN-γ works by several different mechanisms
Adoptive Immunotherapy Adoptive immunotherapies involve the transfer of either cells or antibodies into a host.
These are also referred to as passive therapies, since the host does not actively mount its own immune response
Immunization Prevention of infectious diseases depends on many factors.
Foremost is the presence of a clean water supply, development of sanitary facilities, good nutrition, and good personal hygiene.
More recently, immunization against a particular agent has been the most effective
The best immunity to an agent is achieved by natural infection, which evolves with a clinical or subclinical response to the agent by the host.
problems of live vaccines are generally related to their safety, while the problems of killed vaccines are related mainly to their effectiveness
Live attenuated vaccines are useful because they infect, replicate, and immunize in a manner similar to natural infection but with milder clinical symptoms
Examples include many of the childhood infections such as measles, mumps, and rubella (MMR vaccine), chicken pox (varicella) and Bacille Calmette-Guérin (BCG) for tuberculosis.
Although millions of doses have been administered with no complications, if given to an immunocompromised host (such as primary immunodeficiency or secondary to HIV infection), these live vaccines may cause serious disease.
Killed vaccines consist of suspensions of killed organisms such as typhoid, cholera, and pertussis (although there is now an acellular vaccine) or one of the products or fractions of the organism.
These include toxoids of diphtheria and tetanus and subunits of viruses such as surface hepatitis B antigen. Among the most successful of these types of vaccines has been the use of polysaccharides in the pneumococcal, meningococcal, and Haemophilus influenza vaccines.
In general, the killed vaccines are not as effective as the live viruses because they do not give long-lasting immunity as a live infection does.
most interesting new vaccines has not been developed to eliminate the infectious agent but rather to prevent the development of another far more serious disease – a complication of the initial infection.
This is the Gardisal vaccine manufactured by Merck to protect against human papilloma virus (HPV). HPV infection
The common cytokine receptor γ chain is used by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21
The IL-2R and the IL-15R have a second (β chain) subunit in common as well, but have unique α subunits (see Figure 3.3).
These two cytokine receptors use a common signaling pathway that involves JAK1, JAK3, and STAT5
Thus, the unique feature of IL-2 is to prevent T-cell immune response to self that would lead to autoimmunity. In contrast, IL-15 expression has no effect on regulatory T cells but is an anti-apoptotic factor in several systems.
Furthermore, IL-15 promotes the maintenance of CD8+ CD44 memory T cells. Thus, IL-15 has as its primary role the maintenance of a robust memory response to invading pathogens
The sensitivity of a test is defined as the number of diseased individuals that are positive for the test compared with those who are negative.
The specificity of a test is the proportion of individuals without a given disease that are negative. Thus, a positive test is really restricted to the disease in question
A, Often an animal species is selected for antibody production because it will produce less cross-reactive antibodies to a given tissue.
Larger mammals, such as goats and sheep, are used to obtain larger volumes of serum to be used therapeutically in humans.
A recent fear has been that animals such as sheep or cows may have eaten animal food age contaminated with prion disease.
Thus, polyclonal antibody production for therapeutic uses has often been limited to countries like Australia or New Zealand where there have been no recorded cases of prion disease in mammals
B, In brief, the key to this remarkable advance was the ability to obtain spleen cells from mice that had been immunized with a given antigen and fuse these cells to a non-secreting myeloma cell line, which then produces a single antibody clone when fused with a given B cell present in the spleen cells.
Antibody clones are only produced when the mouse B cell fuses with the myeloma line
Large-scale culture of these antibodies can provide large quantities of antibody that are precise in their reactivity.
Most clinical immunology laboratories rely almost exclusively on these machines, and research labs are also introducing these automated techniques at a rapid pace.
If the concentration of antigen– antibody complex is low, then the immune complexes remain in suspension as fi ne particles, which can disperse a beam of light.
As the complexes increase with concentration of antibody, the complexes will precipitate, and light scattering will decrease.
This degree of dispersion can be measured on a nephelometer
In the radioimmunoassay, one can radiolabel a particular antigen or antibody using either 125I or 14C tagged to the antigen or antibody
The description of ELISAs; (Figure 2.1 top right) is similar to that described for the radioimmunoassay, but in place of the radioactively labeled antibody or antigen, various fluorochromes have been substituted in place of the radioactive label.
Normal C4 levels with low C3 and factor B levels suggest the alternative pathway alone.
Elevation of all three components usually suggests acute or chronic infection.
Acute rheumatic fever is such an example
Assays for immune complexes are best directed toward an analysis of the immune complexes or their deposition in various human disease tissues.
In most cases, the best approach is to receive freshly biopsied nonfrozen material that is then snap frozen and sections cut and stained to test for the presence of appropriate antigen or antibody
Immunoblots
Its beauty is its simplicity and the fact that one can compare different proteins, toxins, and cellular products all at the same time and reach conclusions concerning their commonality or differences or purity
The procedure is relatively simple. The proteins to be studied are run on a standard SDS gel, the percentage of which depends on the known or estimated size of the protein: larger proteins are run in 10 percent gels, while smaller proteins are run on 15 percent gels
The gel is then removed and the proteins in the gel are transferred by another electrical charge to a cellulose membrane.
The membrane is treated overnight with a blocking buffer, washed, and then layered over the membrane with the antibody designed to pick up the binding to the protein (S) in question.
This incubation usually lasts one hour; following washes, the membrane is treated with a species-specifi c second antibody tagged to an enzyme and developed with an enzyme substrate to form a colored band
This technique may be used not only on fresh samples but also in tissues that have formalin-fixed and paraffin-embedded tissues.
In this technique of in situ hybridization, the probes can be applied directly to tissue sections on microscope slides.
However, this technique works only after deparaffi nization and proteolytic digestion are performed to expose intracellular nucleic acid targets.
The probe is detected in the sample either by radiolabeling the probe or radiolabeling an antibody to the probe DNA.
a major revolution in detecting DNA material occurred with the introduction of the PCR assay
This method is particularly valuable since it can markedly amplify a small piece of DNA before cleavage with a restriction enzyme
Complementary oligonucleotide primers from either end of the target DNA are added to the denatured sample, along with a heat-resistant DNA polymerase
The newly synthesized double-stranded DNA is then denatured by heating and exposed again to the polymerase enzyme at a lower temperature
In this way, newly synthesized molecules and original DNA can reassociate with the primer and act as templates for further rounds of DNA synthesis
After completing about thirty cycles (usually two to three hours in an automated machine), the specific target sequence is amplified
more than 1-million-fold. This powerful and sensitive technique can detect a specific DNA sequence from a single cell (e.g., lymphocyte, sperm), fixed pathological specimens, and dried blood spots.
The main disadvantage is that contamination of the reaction mixture with traces of DNA from another source will lead to false positive results.
Thus extreme care in handling specimens to be tested as well as in the test itself is obligatory
MAJOR HISTOCOMPATIBILITY (MHC) ASSAYS
MICROARRAY ASSAYS
